The invention relates to a class of peptide and peptide-like compounds, xe2x80x9cPrevinsxe2x80x9d which inhibit the enzymatic activity of Botulinum toxin B and Tetanus neurotoxins and may be used as molecular building blocks for creating compounds which are optimized for inhibiting the protease activity of Botulinum toxin B and Tetanus neurotoxins.
The Botulinum toxins (Bttxs) are among the most potent toxins to animals, e. g. the LD50 in mice is about 1 ng/kg. Bttxs comprise a family of seven distinct serotypes (A-G). Bttxs are composed of two subunits comprising a 100 kdal nerve-cell targeting heavy chain and a 50 kdal endoproteolytically active light chain. These toxins are Zn-metalloproteases and contain a Zn-protein binding motif HEXXH.
However, Zn-metalloprotease inhibitors, such as angiotensin converting enzyme inhibitors, captopril and phosphoramidon, are not effective inhibitors of Bttxs. Although Zn-chelators inhibit Bttx protease activity in vitro, they merely delay the protease activity in vivo and in tissue preparations comprising intact nerve and muscles cells and/or tissues. Furthermore, some Zn-chelators are toxic at concentrations necessary to delay the Bttx protease activity. Although dithiocarbamates inhibit other Zn-containing proteins such as SOD, they are ineffective against the Bttx serotype B (BttxB). Clearly, inhibitors of the various Bttx serotypes, such as BttxB, are needed.
BttxB specifically cleaves synaptobrevin (VAMP2) between glutamine 76 and phenylalanine 77 (QF bond or cleavage site). There is an obligatory requirement for a relatively long substrate for the in vivo target VAMP2 as shown by efforts to produce a minimum length substrate. It has been shown that 30 amino acids of VAMP2 are required and 40 amino acids of VAMP2 are required for optimum cleavage. See Shone, C. C. et al. (1993) Eur. J. Biochem. 217:965-971. V2, a peptide derived from VAMP2, is a sequence of 10 amino acids located 4 residues upstream from the cleavage site, and was found to inhibit Bttx activity. See Pellizzari R. et al. (1996) J. Biol. Chem. 271:20353-20358. In VAMP2, a mutation of the C-terminal amino acids had little effect, whereas a helix disrupting substitution of Pro for Ala inhibited BttxB activity by 28%. Further, replacement of several negatively charged amino acids led to almost complete inactivity. See Whitcome, M, et al. (1996) FEBS Let. 386:133-136).
Computer-aided secondary structure analysis of VAMP2 predicted two stretches of xcex1-helical structure flanking the cleavage site QF. See Witcome, M. R. et al. (1996) FEBS Let. 386: 133-136. Computer-aided tertiary structure analysis indicates that the two helices could self associate to form a supersecondary structure of a helix bundle with the helices separated by a reverse turn. See Lebeda F. J., et al. (1996) Med. Defense Biosci. Rev. 204.
The above results indicate that more than just the QF bond is required to be recognized by the toxin for substrate cleavage.
The invention is directed to the core structures called xe2x80x9cPrevinsxe2x80x9d which have an internal QF bond and the ability to inhibit BttxB protease activity.
As the tetanus toxin cleavage site is the same as BttxB, the core structures may also serve as the core structures of compounds that competitively inhibit tetanus protease activity.
Thus, in one aspect, the invention is directed to compounds having a structure of the formula:
B1Z*2B3Z*4X*5Q6F7X8X9X10X11xe2x80x83xe2x80x83(1),
B1X2X3X4X5Q6F7X8X9X10X11xe2x80x83xe2x80x83(2),
or
B1X2B3X4Z5Q6F7Z*8X9X10X11xe2x80x83xe2x80x83(3)
and the salts, esters, amides, and acyl forms thereof. Each position represented by a letter indicates a single amino acid residue: B is a basic or polar/large amino acid or a modified form thereof; X is a small or hydrophobic amino acid or a modified form thereof; X* is a small or polar/large amino acid or a modified form thereof; Z is a polar/large or hydrophobic amino acid or a modified form thereof, Z* is Broline or a polar/large or hydrophobic amino acid or a modified form thereof As described below, one or more of the peptide linkages between the amino acid residues may be replaced by a peptide linkage mimic.
In other aspects, the invention is directed to recombinant materials useful for the production of those peptides of the invention that contain gene-encoded amino acids, as well as plants or animals modified to contain expression systems for the production of these peptides. The invention also includes methods to prepare and manipulate these recombinant materials.
In addition, the invention is directed to pharmaceutical compositions containing compounds, containing the core structure of the invention, as active ingredients and to compositions which contain expression systems for the production of the peptides. The invention is also directed to methods to prepare compounds containing the core structure of the invention synthetically, to antibodies specific for these compounds, and to the use of the compounds as preservatives, therapeutics, and prophylactics.
The invention is also directed to the use of the compounds containing the core structures of the invention in assays for detection of BttxB and Tttx by the use of selective inhibition and for determining inhibitors and substrates for a given toxin.
The present invention relates to materials, compositions, kits and methods for inhibiting the enzymatic activity of Botulinum toxin B and Tetanus neurotoxins.
The invention further relates to materials, compositions, kits and methods for preventing or treating toxic poisoning such as Botulinum toxin B and tetanus poisoning. The kits can provide single or multiple dosage and can include other conventional ancillary materials such as instructions, solutions and compositions needed for operation. The compositions and solutions may be placed in containers, test tubes, etc. Containers could be similar to those employed in insect/snake bite kits that includes an injector which provides compounds containing the core structure of the invention and TCEP in separate chambers.
A kit for determining whether a sample contains a compound having the core structure of the invention, the amount of said compound or the type of said compound may include antibodies immunospecific for the core structure.
A kit for determining whether a sample contains a Botulinum toxin or the type of the Botulinum toxin may include antibodies immunospecific for at least one compound containing the core structure having an interaction with a Botulinum toxin. Likewise, a kit for determining whether a sample contains a Tetanus toxin would include antibodies immunospecific for at least one compound containing the core structure of the invention having an interaction with a Tetanus toxin.
Another embodiment includes Buforin I along with one or more known peptide inhibitors associated with the decontamination of Botulinum B and/or Tetanus toxins. Additionally, the kits may also include a stable peptide mixture or powder which includes compounds having the core structures of the invention for sprinkling over food or wounds for detoxification.
Yet another embodiment includes the use of the compounds of the invention as molecular building blocks to construct compounds optimized for inhibition of the protease activity of BttxB and Tttx.